Fusion Expression on the Esat-6 and cfp-10 Genes of Mycobacterium Bovis in Escherichia Coli

摘要

Based on the (Gly4Ser)3 linker, the esat-6 and cfp-10 gene were fused for raising the antigenicity of single antigen. The DNA fragments of esat-6 and cfp-10 were fused by splicing by overlapping extension (SOE) polymerase chain reaction(PCR), and the fusion gene esat-6-cfp-10 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-esat-6-cfp-10. pMD-esat-6-cfp-10 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified mpb esat-6-cfp-10 fusion gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET-esat-6-cfp-10 was constructed. Plasmid containing pET-esat-6-cfp-10 was transformed into competence Escherichia coli BL21(DE3). The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 25 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of DNA vaccine; living carrier vaccine; subunit vaccine and diagnostic reagents against bovine tuberculosis.